A method for detecting interaction between biofilm and biomolecule based on terahertz spectroscopy technology

Abstract

The application discloses a method for detecting biomembrane and biomolecule interaction based on terahertz technology, which comprises the following steps: step 1, preparing a sample containing target biomembrane and target biomolecule; step 2, providing a terahertz spectrum device, wherein a container for containing the sample in step 1 to perform terahertz detection is arranged on the device; and step 3, adding the sample in step 1 into the container in step 2 to perform terahertz spectrum detection, and obtaining optical properties of terahertz frequency of biomembrane and biomolecule interaction. The application provides a method for sensitively detecting biomembrane and biomolecule interaction under near physiological conditions, and the multi-scale motion information carried by the terahertz spectrum can realize detailed characterization of biomembrane and biomolecule interaction at a dynamic level.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for detecting the interaction between biological membranes and biomolecules based on terahertz spectroscopy. Background Technology

[0002] Interactions between biological membranes and biomolecules are widespread in living systems. These interactions lead to functional changes in the relevant biomolecules, thereby affecting the cellular life activities they regulate. In principle, this functional regulation stems from the fact that the overall properties of phospholipid molecules or biological membranes influence the structure and dynamics of macromolecules such as proteins bound to them. For example, binding to the membrane interface allows phospholipase A2 to adopt a more flexible conformation and possesses more favorable kinetic properties for enzyme activity, thus affecting the metabolic pathways in which phospholipase A2 participates. Detecting the interactions between biological membranes and biomolecules and characterizing the structural and kinetic changes in this process in detail not only helps answer key scientific questions in biological membrane research but also inspires the development of new disease therapies targeting membrane proteins, possessing significant scientific and practical implications.

[0003] Current techniques such as surface plasmon resonance (SPR) and electron spin resonance (ESR) used to probe the interaction between biomembranes and biomolecules cannot obtain crucial biological information about structure and dynamics. Structural biology methods, such as X-ray crystallography, are not only expensive but also have stringent requirements regarding sample conditions, making them unsuitable for this research. Developing new methods for probing the interaction between biomembranes and biomolecules is an urgent need in related research fields.

[0004] The search has not yet found any reports of applying terahertz technology to the detection of interactions between biological membranes and biomolecules. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a method for detecting the interaction between biological membranes and biomolecules based on terahertz technology, which can obtain information that is currently unavailable under near-physiological conditions. Terahertz spectral measurements of specially designed biological membrane systems can sensitively detect low-frequency vibrational changes generated by the binding of biomolecules to the membrane, thereby elucidating the subtle dynamic evolution in this process.

[0006] The objectives of this invention and the solutions to its technical problems can be achieved by the following technical solutions.

[0007] In a first aspect, the present invention first protects a method for detecting the interaction between biological membranes and biomolecules based on terahertz technology, comprising the following steps:

[0008] Step 1: Prepare a sample containing the target biomembrane and the target biomolecule;

[0009] Step 2: Provide a terahertz spectroscopy device, which is equipped with a container for holding the sample described in Step 1 for terahertz detection;

[0010] Step 3: Add the sample described in Step 1 to the container described in Step 2, and perform terahertz spectroscopy detection to obtain the optical properties of the terahertz frequency of the interaction between the biological membrane and biomolecules.

[0011] The biomembrane described in step 1 is a self-assembled structure formed by lipid molecules in an aqueous solution, with the target molecules interacting with the biomembrane dissolved in the aqueous solution. The self-assembled structure can be any biomembrane structure, including solid support membranes, water-in-oil reverse micelle structures, liposomes, etc. These self-assembled structures are ideal model biomembrane systems, referred to by researchers both domestically and internationally as "artificial cell" structures. Compared to the crystal samples used in the structural biology methods described in the background section, these model biomembrane systems can retain an aquatic environment similar to real physiological regulation and can simulate many characteristics of the micro-mesoenvironment of biomembranes in cells, providing a suitable platform for exploring the interactions between biomembranes and biomolecules during physiological processes and their structural and dynamic changes.

[0012] The terahertz spectroscopy device mentioned in step 2 is a terahertz time-domain spectrometer or a Fourier transform infrared spectrometer capable of providing terahertz frequency band spectral measurements, and its sample detection mode is transmission mode, reflection mode, or attenuated total internal reflection mode. Depending on the sample detection mode used, the container mentioned in step 2 is an optical cuvette or an attenuated total internal reflection prism. According to a preferred embodiment of this application, the optical cuvette is made of a material with good transmittance in the terahertz band, such as quartz.

[0013] The optical properties of the terahertz frequencies mentioned in step 3 refer to the changes in optical parameters resulting from the interaction between the biomembrane and biomolecules in the sample. These optical parameters include absorption coefficient, complex refractive index, and complex dielectric constant, and the spectral information they carry reveals dynamic processes such as the collective vibration of biomolecules and the hydrogen bond network vibration of water molecules at the biomembrane interface. These terahertz frequency movements are closely related to biological processes such as conformational changes in biomolecules, mass transfer at the membrane interface, and biochemical reactions involving water, and are considered to have significant functional importance. Therefore, this application applies terahertz spectroscopy to the study of interactions between biomembranes and biomolecules, focusing on the subtle changes in terahertz spectra generated during the interaction process, thereby characterizing the evolution of the aforementioned dynamic processes between the biomembrane interface and biomolecules during this process.

[0014] As a preferred technical solution of this application, the biomolecules mentioned in step 1 are one or more of ions, polypeptides, proteins, nucleic acids, and carbohydrates.

[0015] As a preferred technical solution of this application, the main component of the biomembrane in step 1 is lipids, including glycerophospholipids, sphingomyelins, steroids, etc.; the secondary components are proteins or carbohydrates, which are selectively added according to the actual needs of the detection.

[0016] Preferably, the glycerophospholipid is one or more of phosphatidylcholine, phosphatidylglycerol, and phosphatidylserine; the glycerophospholipid may be phosphatidylcholine, phosphatidylglycerol, or phosphatidylserine alone, or it may be a mixture of phosphatidylcholine and phosphatidylglycerol, phosphatidylcholine and phosphatidylserine, phosphatidylglycerol and phosphatidylserine, or phosphatidylcholine, phosphatidylglycerol, and phosphatidylserine in a certain proportion.

[0017] It should be understood that, depending on the detection mode of the terahertz spectrometer, different types of biomembrane structures can be selected to obtain optimal detection sensitivity. For example, when the detection mode is transmission mode, biomembrane samples in the form of water-in-oil reverse micelles or liposomes can be selected; when the detection mode is attenuated total reflection mode, biomembrane samples in the form of solid supported membranes can be selected.

[0018] As a preferred embodiment of this application, the biomembrane structure is a water-in-oil reverse micelle. The reverse micelle contains an aqueous phase with target biomolecules that interact with the biomembrane, and an oil phase on the outside. The oil phase is a reagent containing dissolved lipid molecules and exhibiting good optical transmittance in the terahertz frequency range, such as hexadecane. This type of biomembrane structure provides an aqueous environment similar to that inside a cell or membrane-containing cell structure, representing one of the most classic biomembrane models. Samples in the form of water-in-oil reverse micelles can confine target biomolecules within a micro-nanoscale aqueous phase inside the reverse micelles, reducing signal interference from irrelevant water molecules and facilitating highly sensitive terahertz spectroscopy detection.

[0019] Preferably, the preparation of the reverse micelle biofilm sample includes the following steps:

[0020] 1) Dissolve the target biomolecule in a buffer solution to serve as the aqueous phase of the reverse micelle emulsion;

[0021] 2) All lipids used to form biological membranes are dissolved in organic reagents to form the oil phase of the reverse micelle emulsion;

[0022] 3) The aqueous phase is added to the oil phase, and mechanical stress is applied to obtain a uniform and stable water-in-oil emulsion sample.

[0023] Preferably, the mechanical stress is generated by one or more of the following methods: vortexing, stirring, and ultrasound.

[0024] In one specific embodiment, the volume ratio of the oil phase to the water phase can be 4:1, 9:1, 19:1, etc., preferably 19:1.

[0025] In a specific embodiment, the calculation method for each optical parameter is as follows:

[0026] For transmission mode measurements, the absorption coefficient α(ν) at frequency ν is calculated based on the sample detection cavity length d, the terahertz signal intensity Ir(ν) of the empty cuvette, and the terahertz signal intensity Is(ν) transmitted through the sample:

[0027] α(ν)=d -1 ln[I r (ν) / I s (ν)];

[0028] The refractive index n(ν) is calculated based on the terahertz signal phase Φr(ω) passing through the empty cuvette and the terahertz signal phase Φs(ω) transmitted through the sample:

[0029] n(ν)=n r (ν)+c(2πνd) -1 (φ s (ν)-φ r (ν));

[0030] The imaginary part of the refractive index, k(ν), is calculated based on the following formula:

[0031] κ(ν)=α(ν) / 2πν;

[0032] The real part ε'(ν) and the imaginary part ε'(ν) of the dielectric constant are calculated based on the following formula:

[0033] ε'(ν)=n 2 (ν)-κ 2 (ν);

[0034] ε"(ν)=2n(ν)·κ(ν).

[0035] For reflection mode measurements, the complex permittivity of the sample Fresnel reflectance based on prism-sample layer Calculation of the terahertz signal incident angle θ and the complex permittivity ε1 of the ATR prism:

[0036]

[0037] Among them, the Fresnel reflectance coefficient of the prism-sample layer Based on the detected reflectance Phase spectrum Fresnel reflectance of prism-air layer calculate:

[0038]

[0039]

[0040] As a preferred embodiment of this application, the biomembrane is a solid-supported membrane, which is a bilayer or multilayer biomembrane deposited on a solid carrier. The upper part of the bilayer or multilayer biomembrane contains an aqueous solution containing target biomolecules that interact with the biomembrane. When using the attenuated total internal reflection mode for detection, the solid carrier of the biomembrane serves as an attenuated total internal reflection prism. The aqueous environment of this type of biomembrane interface is similar to the surface of a cell membrane with low curvature or a membrane-containing cell structure, and is one of the most classic biomembrane models. The combination of a solid-supported membrane sample with the attenuated total internal reflection detection mode can maximize the highlighting of the optical properties at the membrane interface, which is beneficial for high-sensitivity terahertz spectroscopy detection.

[0041] Preferably, the preparation of the solid supported film sample includes the following steps:

[0042] 1) Dissolve the target biomolecule in a buffer solution to form an aqueous solution;

[0043] 2) Clean the attenuating total reflection prism and prepare a solid support membrane on the attenuating total reflection prism according to the requirements of the number of biofilm layers required for the experiment.

[0044] 3) The aqueous solution containing the target biomolecule is dropped onto the solid support membrane and incubated for a period of time as required by the experiment to obtain a solid support membrane sample containing the membrane structure and the target biomolecule.

[0045] Preferably, the method for preparing the solid support film on the attenuating total reflection prism is one of the following: Langmuir-Blodgett (LB) impregnation method, vesicle adsorption fusion method, or LB method combined with vesicle fusion method.

[0046] To further improve the sensitivity of the detection, the terahertz spectrometer is also equipped with a terahertz metamaterial sensor.

[0047] Preferably, the terahertz material sensor is made of terahertz metamaterial.

[0048] More preferably, the terahertz metamaterial is engraved with a periodic structure of Asia-Pacific terahertz wavelength, such as a double-strip structure.

[0049] When using terahertz metamaterial sensors, the optical properties of the terahertz frequencies detected by this invention can also include the amplitude and frequency variations of the resonance peaks.

[0050] As a preferred technical solution of this application, the container described in step 2 is an optical cuvette or an attenuating total internal reflection prism. It should be understood that the type of container used is determined according to the sample detection mode of the terahertz spectrometer. When using transmission or reflection mode, the container is an optical cuvette; when using attenuating total internal reflection mode, the container is an attenuating total internal reflection prism. The optical cuvette is made of a material with good transmittance in the terahertz band and good compatibility with the components of the sample; preferably, it is quartz.

[0051] This invention, for the first time, creatively proposes to detect and describe key changes in the interaction process between biomembranes and biomolecules based on structural dynamics and hydration dynamics information revealed by terahertz spectroscopy. Compared with existing technologies, this invention has at least the following beneficial technical effects:

[0052] 1) It has no strict requirements on the sample form and can achieve detection under near-physiological conditions;

[0053] 2) No additional probes are required, resulting in lower detection costs;

[0054] 3) The information obtained is abundant and has significant biological value;

[0055] 4) Applicable to the detection of various strong or weak interactions, with a wide range of applications;

[0056] 5) It can be combined with terahertz metamaterials, resulting in high detection sensitivity;

[0057] 6) In theory, it is possible to detect multiple interactions simultaneously. Attached Figure Description

[0058] Figure 1 This is a schematic diagram illustrating the detection of the interaction between biological membranes and biomolecules according to the present invention;

[0059] Figure 2 This is a schematic diagram of a transmission mode terahertz time-domain spectrometer according to an embodiment of the present invention;

[0060] Figure 3 This is a schematic diagram illustrating the detection of the interaction between phosphatidylglycerol and metallic copper ions according to Embodiment 1 of the present invention;

[0061] Figure 4 This is a terahertz absorption spectrum for detecting the interaction between phosphatidylglycerol and metallic copper ions according to Example 1 of the present invention;

[0062] Figure 5 This is a schematic diagram of a terahertz spectroscopy device in attenuated total reflection mode according to an embodiment of the present invention;

[0063] Figure 6This is a terahertz absorption spectrum for detecting the interaction between phospholipase A2 and a phosphatidylserine-containing solid support membrane according to Example 2 of the present invention;

[0064] Figure 7 This is a terahertz absorption spectrum for detecting the interaction between phospholipase A2 and a phosphatidylserine-containing reverse micelle membrane, according to Example 3 of the present invention;

[0065] Figure 8 This invention relates to Example 4 of the present invention, which detects the terahertz absorption spectrum and the imaginary part spectrum of the dielectric constant of the interaction between coagulation factor VIII protein and phosphatidylserine-containing membrane;

[0066] Figure 9 This is a schematic diagram of a dual-strip metamaterial sensor according to Embodiment 5 of the present invention;

[0067] Figure 10 This is a schematic diagram of the detection of the interaction between multiple mixed metal ions and multi-component biological membranes using terahertz metamaterials according to Embodiment 5 of the present invention;

[0068] Figure 11 According to Embodiment 5 of the present invention, terahertz metamaterials are used to detect the terahertz absorption spectra of phosphatidylserine and phosphatidylglycerol membranes.

[0069] Figure 12 According to Embodiment 5 of the present invention, terahertz metamaterials are used to detect the terahertz absorption spectra of two metal ions in a biomembrane system containing different proportions of phosphatidylserine and phosphatidylglycerol.

[0070] Figure 13 This is the terahertz absorption spectrum of the present invention, obtained by combining terahertz metamaterials to detect the interaction between various mixed metal ions and multi-component biological membranes according to Embodiment 5 of the present invention.

[0071] Figure 14 This is a schematic diagram illustrating the detection of complex interactions between multi-component biomembranes and biomolecules such as ions and proteins in artificial cells according to Embodiment 6 of the present invention.

[0072] Figure 15 This is a terahertz absorption spectrum obtained by detecting the complex interactions between multi-component biological membranes and biomolecules such as ions and proteins in artificial cells according to Embodiment 6 of the present invention.

[0073] in:

[0074] 1-Biomolecule; 2-Biological membrane; 3-Lipid components interacting with biomolecules; 4-Femtosecond laser; 5-Spectrometer; 6-Pump beam; 7-Detector beam; 8-Emitter; 9-Time delay device; 10-Receiver; 11-Lock-in amplifier; 12-Sample detection chamber; 13-Hexadecane; 14-Glycerophospholipid; 15-Copper ion; 16-Ion-free phosphatidylcholine sample; 17-Ion-free phosphatidylglycerol sample; 18-Ion-free phosphatidylserine sample; 19-Copper-containing phosphatidylcholine sample; 20-Copper-containing phosphatidylglycerol sample; 21-Copper-containing phosphatidylserine sample; 22-Signal transmitter; 23-Signal receiver; 24-Attenuating total internal reflection prism; 25-Solid support membrane sample; 26-0 minute after addition of phospholipase; 27- 30 minutes after adding phospholipase; 28 - Differential spectrum; 29 - Sample containing 5% phosphatidylserine; 30 - Sample containing 10% phosphatidylserine; 31 - Sample containing 20% ​​phosphatidylserine; 32 - Sample without phosphatidylserine; 33 - Sample containing 5% phosphatidylserine; 34 - Sample containing 25% phosphatidylserine; 35 - Phosphatidylserine micelles; 36 - Phosphatidylglycerol micelles; 37 - Phosphatidylserine:phosphatidylglycerol = 1:3; 38 - Phosphatidylserine:phosphatidylglycerol = 3:1; 39 - Sample containing 100% copper ions; 40 - Sample containing 100% zinc ions; 41 - Sample containing 50% copper ions and 50% zinc ions; 42 - Sample containing 100% copper ions; 43 - Sample containing 100% zinc ions; 44 - Sample containing 50% copper ions and 50% zinc ions. Detailed Implementation

[0075] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art should understand that the embodiments described below are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0076] This invention provides a method for detecting the interaction between biomembranes and biomolecules based on terahertz technology, and a biomembrane detection system for detecting the interaction between biomembranes and biomolecules, such as... Figure 1 As shown, the biomembrane can be any form of biomembrane surface, with biomolecules located in the aqueous phase, interacting with specific components on the biomembrane, such as specific lipid molecules. Preferably, the biomembrane is in the form of a water-in-oil reverse micelle or a solid supported membrane. This invention can sensitively detect changes in terahertz frequency vibrations in the detection system caused by the interaction between the biomembrane and biomolecules by utilizing changes in various optical properties obtained through terahertz spectroscopy, thereby analyzing information on the structural and dynamic changes of biomolecules during the interaction process.

[0077] The preparation method of the aforementioned water-in-oil reverse micelle detection system is as follows: glycerol phospholipids dissolved in an organic solvent such as chloroform are dried with nitrogen gas, and the solvent is removed by vacuum to form a thin film. The thin film phospholipids are dissolved in hexadecane to obtain an oil phase. If necessary, biomolecules are dissolved in ultrapure water / buffer (such as TBS) to obtain an aqueous phase. Then, the oil phase and the aqueous phase are mixed at a volume ratio of 19:1, and a stable emulsion sample is formed by vortex sonication.

[0078] The preparation method of the aforementioned solid support membrane detection system is as follows: Glycerol phospholipids dissolved in an organic solvent such as chloroform are dried with nitrogen gas, and the solvent is removed by vacuum to form a thin film. The thin film phospholipids are dispersed in ultrapure water / buffer (such as TBS) and ultrasonically sonicated to obtain a liposome solution. After cleaning and hydrophilic treatment of the target substrate, the liposome solution is dropped onto the substrate surface, and a humid environment is maintained. The substrate is incubated at a temperature higher than the phospholipid phase transition temperature for at least 0.5 hours. After incubation, the phospholipid membrane surface is washed with ultrapure water / buffer (such as TBS) to obtain a solid support membrane composed of bilayer phospholipid molecules.

[0079] Preferably, when the biomolecules are ions (such as copper ions, zinc ions, sodium ions, etc.), they can be directly dissolved in pure water. When the biomolecules are polypeptides, proteins, nucleic acids, sugars, etc., a buffer solution such as TBS is selected to dissolve the biomolecules in order to improve the stability of the aqueous phase.

[0080] The preferred embodiments of the present invention will now be described in detail with reference to examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and intent, and all such modifications and substitutions fall within the scope of protection claimed in the present invention.

[0081] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0082] Example 1: Detection of the interaction between copper ions and phosphatidylglycerol

[0083] Example 1 illustrates the application of the method of this invention to detect the interaction between copper ions and phosphatidylglycerol. The biomembrane system detected in this example is a water-in-oil reverse micelle, such as... Figure 3As shown, its composition is as follows: The membrane component is glycerophospholipid 14, which is one of phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol. It self-assembles at the oil-water interface between hexadecane 13 and the aqueous phase to form a biomembrane structure, forming a water-in-oil reverse micelle sample. The aqueous phase is pure water dissolved with a concentration of 10 mM copper ions 15. Specifically, the sample preparation method is as follows: 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine, 1,2-dioleoyl-sn-glycerol-3-phosphatidylserine, or 1,2-dioleoyl-sn-glycerol-3-phosphatidylglycerol dissolved in chloroform is dried with nitrogen gas, and the solvent is removed by vacuum to form a thin film. The thin film phospholipid is dissolved in hexadecane to obtain the oil phase; copper chloride solid is dissolved in ultrapure water to obtain the aqueous phase; the oil phase and the aqueous phase are mixed at a volume ratio of 19:1, and a stable emulsion sample is formed by vortex sonication.

[0084] In this embodiment, the following is used: Figure 2 The terahertz time-domain spectroscopy device is used to test the sample. The terahertz pulse generated by the femtosecond laser 4 is split into a pump beam 6 and a probe beam 7 by a beam splitter 5. The pump beam 6 passes through the emitter 8 and then through the quartz sample detection cavity 12. The probe beam 7 uses a time delay device 9 to adjust the time delay between the pump pulse and the probe pulse. The signal is received by the receiver 10, and after passing through the lock-in amplifier 11, the phase and amplitude of the signal are measured. Finally, the entire time-domain waveform of the terahertz pulse can be detected. By Fourier transform, the frequency domain spectrum of the sample under test can be obtained, thereby obtaining its absorption coefficient, refractive index, transmittance and other optical parameters.

[0085] The calculation methods for the various optical parameters measured in this embodiment are as follows, wherein...

[0086] The absorption coefficient α(ν) at frequency ν is calculated based on the sample detection cavity length d, the terahertz signal intensity Ir(ν) of the empty cuvette, and the terahertz signal intensity Is(ν) transmitted through the sample:

[0087] α(ν)=d -1 ln[I r (ν) / I s (ν)];

[0088] The refractive index n(ν) is calculated based on the terahertz signal phase Φr(ω) passing through the empty cuvette and the terahertz signal phase Φs(ω) transmitted through the sample:

[0089] n(ν)=n r (ν)+c(2πνd) -1 (φ s (ν)-φ r (ν));

[0090] The imaginary part of the refractive index, k(ν), is calculated based on the following formula:

[0091] κ(ν)=α(ν) / 2πν;

[0092] The real part ε'(ν) and the imaginary part ε'(ν) of the dielectric constant are calculated based on the following formula:

[0093] ε'(ν)=n 2 (ν)-κ 2 (ν);

[0094] ε"(ν)=2n(ν)·κ(ν).

[0095] In this embodiment, the terahertz spectrum of the blank biomembrane system without copper ions was first detected. Figure 4 In the frequency range shown in the spectra (0.4-1.8 THz), the terahertz absorption spectra mainly reflect the differences in the hydration state of the aqueous phase. The terahertz spectra of biofilm samples 16, 17, and 18 prepared from phosphatidylcholine, phosphatidylglycerol, and phosphatidylserine showed no significant differences within the indicated frequency range, indicating that in the current detection system, the different types of phospholipids do not produce changes in hydration properties sufficient to significantly affect the terahertz spectra. Observation of the terahertz spectra of samples 19, 20, and 21 corresponding to the addition of copper ions to the three phospholipids revealed that the absorption coefficients all increased after the addition of ions. This change should be attributed to the sum of the absorption of the metal ions themselves and the perturbation of the hydration state by the metal ions. Specifically, the absorption coefficients of the phosphatidylcholine-copper ion sample and the phosphatidylserine-copper ion sample were similar, while the absorption coefficient of the phosphatidylglycerol-copper ion sample was significantly higher than that of the other two phospholipid-copper ion samples. The spectral results of the blank group and the samples containing phosphatidylcholine, phosphatidylserine, and copper ions have demonstrated that the absorption of phospholipids and copper ions themselves, as well as the absorption resulting from changes in hydration state, are consistent or show no significant differences in each group. Therefore, it is reasonable to infer that the higher absorption coefficient of the phosphatidylglycerol-copper ion sample originates from the different interactions between the components in the system. In the three experimental groups, copper ions (considering the difficulty in binding between the negative charge on the biological membrane and the negatively charged chloride ions, this interaction should be an interaction between the phospholipid membrane and copper ions) may bind to the phospholipid membrane, thereby causing changes in hydration state and affecting the terahertz spectrum. The degree of this effect is determined by the affinity between the membrane and the ions. Based on the spectral results, it can be inferred that copper ions tend to bind more readily to phosphatidylglycerol. This inference is consistent with reports in domestic and international literature on the high affinity between copper ions and phosphatidylglycerol. This preliminarily demonstrates that this experimental system specifically detects the interaction between phosphatidylglycerol and copper ions.

[0096] Example 2: Detection of the interaction between phospholipase A2 and phosphatidylserine-containing solid support membrane

[0097] According to the method of the present invention, the detection of the interaction between phospholipase A2 and a phosphatidylserine-containing solid support membrane is presented as Example 2. The biomembrane system detected in this example is a solid support membrane, such as... Figure 5 As shown, its composition is as follows: the lipid component of the solid support membrane 25 being detected is glycerophospholipid, specifically composed of two phospholipid molecules: phosphatidylcholine and phosphatidylserine, with phosphatidylcholine comprising 95% by mass and phosphatidylserine comprising 5% by mass. The detection mode used is attenuated total internal reflection mode, and the carrier of the solid support membrane 25 is an attenuated total internal reflection prism 24. Optical paths 22 and 23 are shown as the incident and receiving detection devices for terahertz waves, respectively. The upper part of the supporting solid membrane 25 contains a tris(hydroxymethyl)aminomethane hydrochloride buffer (TBS) at pH 7.4, containing a concentration of 0.5 mg / ml porcine pancreatic phospholipase A2. Specifically, the sample preparation method is as follows: 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine and 1,2-dioleoyl-sn-glycerol-3-phosphatidylserine dissolved in chloroform are mixed at a mass ratio of 95:5, dried with nitrogen, and the solvent is removed by vacuum to form a thin film. The thin film phospholipid is dispersed in TBS buffer and diluted to a lipid concentration of 0.2 mM. The solution is obtained by sonication. A clean silicon attenuating total internal reflection prism is pretreated with sphagnum molybdenum acid (sulfuric acid: hydrogen peroxide = 4:1) for 1 h and then washed. 20 μL of the liposome solution is dropped onto the cleaned attenuating total internal reflection prism surface and incubated in a humid environment for 0.5 h. After incubation, the resulting membrane structure is washed with buffer to obtain a solid support membrane. The porcine pancreatic phospholipase A2 solution is dropped onto the solid support membrane to obtain the detection system.

[0098] In this embodiment, the spectral system and optical parameter calculation methods used employ a commercial Fourier transform infrared spectrometer and the commercial software OMNIC from Thermo Fisher Scientific. In this embodiment, as... Figure 6As shown, terahertz spectra were measured 0 minutes and 30 minutes after the addition of phospholipase A2 solution (26, 27). To compare the differences, the difference spectrum (28) was calculated. Clearly, the difference spectrum (28) shows numerous distinct spectral details in the 18-120 THz range, such as the absorption peak near 50 THz. This spectral information reflects changes in vibrational information, such as the stretching vibration of water molecules' hydroxyl groups. After the addition of phospholipase A2 solution, the macroscopic composition of the system remains unchanged, but phospholipase A2, as a membrane protein, gradually interacts with the phospholipid membrane, i.e., it gradually adsorbs onto the solid support membrane. This process is time-varying; the proportion of phospholipase A2 adsorbed onto the phospholipid membrane increases with time. The changes in spectral information reflect the changes in vibrational information at terahertz frequencies during this process. Furthermore, if other frequency bands (such as 0.1-18 THz) are required, this can be achieved by changing the detection range of the detector in a commercial Fourier transform infrared spectrometer; the experimental method is consistent with this embodiment. The results preliminarily demonstrate that the method proposed in this invention can sensitively detect the interaction between phospholipase A2 and a solid support membrane containing phosphatidylserine, and obtain the subtle changes in hydration dynamics and conformational dynamics during this process.

[0099] Example 3: Detection of the interaction between phospholipase A2 and phosphatidylserine-containing reverse micelle membranes

[0100] According to the method of the present invention, the interaction between phospholipase A2 and a phosphatidylserine-containing reverse micelle membrane was detected as Example 3. The biomembrane system detected in this example was a reverse micelle membrane, the composition of which was as follows: the lipid component of the membrane consisted of two phospholipid molecules, phosphatidylcholine and phosphatidylserine, which self-assembled at the oil-water interface of hexadecane and aqueous phase to form a biomembrane structure, forming a water-in-oil reverse micelle sample. The aqueous phase was a tris(hydroxymethyl)aminomethane hydrochloride buffer (TBS) at pH 7.4, containing 0.5 mg / ml porcine pancreatic phospholipase A2. Specifically, the sample preparation method is as follows: 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine and 1,2-dioleoyl-sn-glycerol-3-phosphatidylserine dissolved in chloroform are dried with nitrogen gas, and the solvent is removed by vacuum to form a thin film. The thin film phospholipid is dissolved in hexadecane to obtain the oil phase. Phospholipase A2 solid from porcine pancreas is dissolved in TBS to obtain the aqueous phase. The oil phase and aqueous phase are mixed at a volume ratio of 19:1 and a stable emulsion sample is formed by vortex sonication.

[0101] In this embodiment, the spectral system and optical parameter calculation method used are as described in Embodiment 1. In this embodiment, as... Figure 7As shown, the terahertz spectra of the interaction between phosphatidylcholine / phosphatidylserine mixed biomembranes with different phosphatidylserine contents and phospholipase A2 were detected. The proportions of phosphatidylserine in samples 29, 30, and 31 were 5%, 10%, and 20%, respectively. The results showed that the intensity of the terahertz absorption spectrum increased with the increase of the phosphatidylserine content. The results of Example 1 have shown that, in the absence of protein, changes in phospholipid types do not produce significant differences in terahertz spectral signals, while the concentration of phospholipase A2 is consistent in all groups, and the two independent components do not produce... Figure 7 The differences in absorption spectra shown suggest that the spectral differences originate from the varying interactions between the two components: the phospholipid membrane and phospholipase A2. Biomembranes with different phosphatidylserine contents can bind to phospholipase A2, causing changes in the free water / hydrated water ratio and the low-frequency vibrations of phospholipase A2, thus contributing to the terahertz spectrum. Therefore, differences in binding affinity or binding mode can produce differences in terahertz spectra. When the phosphatidylserine content increases, the negative charge on the membrane increases, which favors the interaction between the positively charged amino acids on phospholipase A2 and the membrane; this has been confirmed by relevant studies both domestically and internationally. Based on this inference, the differences in terahertz spectra reflect the different modes or affinities of interaction between phospholipid membranes with different compositions and phospholipase A2. These results preliminarily demonstrate that the method proposed in this invention can sensitively detect the interaction between phospholipase A2 and phosphatidylserine-containing membranes, as well as the subtle changes in hydration kinetics and conformational kinetics during this process.

[0102] Example 4: Detection of the interaction between coagulation factor VIII protein and phosphatidylserine-containing membranes

[0103] According to the method of the present invention, the interaction between coagulation factor VIII protein and phosphatidylserine-containing membrane is used as Example 4. Compared with the phospholipase A2 protein detected in Example 3, the molecular weight of coagulation factor VIII protein is significantly larger, and its interaction with the membrane is more complex, which is beneficial for analyzing the feasibility of the method proposed in this invention when analyzing proteins with large differences. The biomembrane system detected is similar to that in Example 3, and its composition is as follows: the lipid component of the membrane is two phospholipid molecules, phosphatidylcholine and phosphatidylserine, which self-assemble at the oil-water interface of hexadecane and aqueous phase to form a biomembrane structure, forming a sample in the form of water-in-oil reverse micelles. The aqueous phase is tris(hydroxymethyl)aminomethane hydrochloride buffer (TBS) with a concentration of 0.1 mg / ml coagulation factor VIII protein and a pH of 7.4. Specifically, the sample preparation method is as follows: 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine and 1,2-dioleoyl-sn-glycerol-3-phosphatidylserine dissolved in chloroform are dried with nitrogen gas, and the solvent is removed by vacuum to form a thin film. The thin film phospholipid is dissolved in hexadecane to obtain the oil phase; the coagulation factor VIII protein solid is dissolved in TBS to obtain the aqueous phase; the oil phase and the aqueous phase are mixed at a volume ratio of 19:1, and a stable emulsion sample is formed by vortex sonication.

[0104] In this embodiment, the spectral system and optical parameter calculation method used are as described in Embodiment 1. Figure 8 As shown, the absorption spectra and imaginary part spectra of the interaction between phosphatidylcholine / phosphatidylserine mixed biomembranes 32, 33, and 34 with coagulation factor VIII protein were detected, with phosphatidylserine contents of 0%, 5%, and 25%, respectively. The results showed that the intensity of the terahertz absorption spectrum significantly increased with the increase of the phosphatidylserine content. This result is similar to the detection results in Example 2, and similar to the analysis in Example 3, it can be reasonably inferred that the spectral differences originate from the different interactions between the phospholipid membrane and coagulation factor VIII. The difference in interaction is due to the change in the charge properties of the phospholipid membrane, and the change in interaction alters the hydration state and low-frequency vibrations of the protein in the system, which is the reason for the change in the dielectric properties at the terahertz frequency. This result is consistent with the findings reported by relevant domestic and international research that negatively charged phospholipids are beneficial to the binding of coagulation factor VIII protein to the membrane. These results, along with those in Example 2, indicate that the method proposed in this invention can be widely used for the interaction of various proteins with biomembranes, and has broad application prospects in the field of membrane protein science and the development of drugs targeting membrane proteins.

[0105] Example 5: Detecting the interaction between various mixed metal ions and multi-component biomembranes using terahertz metamaterials.

[0106] The method of the present invention combines terahertz metamaterials to detect the interaction between multiple mixed metal ions and multi-component biofilms as Example 5. For complex systems containing multiple metal ions and multi-component biofilms, diverse biofilm-metal ion interactions may exist, thus requiring higher detection sensitivity. In this example, the method combines... Figure 9 The dual-strip terahertz metamaterial shown was used for high-sensitivity detection. The composition of the biomembrane system being detected is as follows: the lipid component of the membrane consists of three phospholipid molecules: phosphatidylcholine, phosphatidylglycerol, and phosphatidylserine (of which phosphatidylcholine accounts for 60% of the total mass, and the remaining 40% is phosphatidylglycerol and phosphatidylserine in adjustable proportions). These molecules self-assemble at the oil-water interface between hexadecane and the aqueous phase to form a biomembrane structure, forming a sample in the form of water-in-oil reverse micelles. The aqueous phase is pure water dissolved with a total concentration of 100 μM copper and zinc ions. Specifically, the sample preparation method is as follows: 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine, acyl-sn-glycerol-3-phosphatidylglycerol, and 1,2-dioleoyl-sn-glycerol-3-phosphatidylserine dissolved in chloroform are dried with nitrogen gas, and the solvent is removed by vacuum to form a thin film. The thin film phospholipid is dissolved in hexadecane to obtain the oil phase. Copper chloride and zinc chloride solids are dissolved in ultrapure water to obtain the aqueous phase. The oil phase and aqueous phase are mixed at a volume ratio of 19:1, and a stable emulsion sample is formed by vortex sonication.

[0107] In this embodiment, the spectral system and optical parameter calculation method used are as described in Example 1. The fabrication of this dual-strip terahertz metamaterial is prior art; however, it is provided here as an example, not a limitation, of the following fabrication method: 1) Mask fabrication. A structural diagram of the device surface is drawn, marking the structures to be etched. An electron beam exposure is used to fabricate a mask on a chromium plate according to the structural diagram. After exposure, the areas to be retained will become shadowed, while the areas to be etched will remain transparent. 2) Material cleaning. The mask and the quartz sheet used to prepare the metamaterial are cleaned with a sulfuric acid and hydrogen peroxide cleaning solution. 3) Photoresist application. Photoresist is generally used as a medium for transferring optical exposure, electron beam, or ion beam lithography patterns. In this process, positive photoresist is used, meaning that the photosensitive compound dissolves in the developer after exposure, while the unexposed areas do not dissolve. The photoresist is coated onto a quartz wafer (500 μm thick) using a spin coater. First, hexamethyldisilazane is spin-coated as an adhesion promoter to increase the adhesion between the wafer and the photoresist. Then, the photoresist is coated, with the coating thickness controlled by the spin coater's speed and spin time. Finally, the sample is dried to remove the solvent from the photoresist. 4) UV Exposure: The photoresist-coated quartz wafer is pressed under a photomask for UV exposure. 5) Development: The sample is immersed in tetramethylammonium hydroxide developer. The exposed photoresist dissolves in the developer, while the unexposed portions remain coated on the sample surface. The sample is then baked in a dryer to evaporate any remaining developer. 6) Metal Plating: First, a 10 nm thick chromium layer is deposited on the substrate as an adhesion layer, followed by a gold film approximately 50 nm thick. 7) Photoresist Removal: Oxygen is used as the removal gas. Under high voltage, oxygen ionizes to generate oxygen ions, oxidizing the photoresist into volatile gases, which are then removed by a vacuum pump. 8) Slicing. The processed sample is slicing with a laser to cut into small pieces smaller than the terahertz detection cavity. At this point, the fabrication of the terahertz metamaterial is complete.

[0108] The biofilm system detected in this embodiment is as follows: Figure 10 As shown, possible interactions exist between different metal ions and various phospholipid components of biofilms. Since the binding of metal ions to the biofilm surface alters the hydration kinetics at the membrane interface, this causes changes in the real and imaginary parts of the dielectric constant within the terahertz frequency range. This change is ultimately reflected in the intensity changes and frequency shifts of the terahertz metamaterial resonance peak. The detection results of Example 1 of this invention have demonstrated that the terahertz spectrum of ion-containing reverse micelles is less affected by the type of phospholipid, such as... Figure 11 As shown, this is further demonstrated in metamaterial systems, where the terahertz spectra of ion-free phosphatidylserine micelles 35 and phosphatidylglycerol micelles 36 are very close. This embodiment further investigated the terahertz spectra of samples 37 and 38 with micellar phospholipid compositions of phosphatidylserine:phosphatidylglycerol = 1:3 and 3:1 after the addition of different ions. Figure 12As shown, the left and right figures are the terahertz spectra of micelles containing 100 μM copper and zinc ions, respectively. The results show that the frequency and intensity of the metamaterial's resonance peaks vary significantly with different phospholipid compositions. In particular, copper ions exhibit higher resonance peak amplitudes in film systems with higher PG content, and zinc ions exhibit higher absorption coefficients in film systems with higher PS content. The ion concentrations are consistent across groups with different phospholipid compositions, consistent with the analysis in Example 1. Figure 11 The ion-free spectrum reveals that this spectral change is due to additional signals generated by the interaction between metal ions and phospholipid membranes, rather than signals resulting from compositional changes. The different interactions between phospholipid membranes with different compositions and metal ions are due to the varying affinities of the interactions between different ions and phospholipid molecules. This result preliminarily demonstrates that the method proposed in this invention can be used to obtain terahertz signals generated by various metal ion-phospholipid membrane interactions and provides hydration state information carried by the terahertz spectrum for further analysis.

[0109] In this embodiment, the terahertz spectrum of mixed metal ions in a multi-component phospholipid membrane system was further investigated. For example... Figure 13 As shown, terahertz spectra were detected when the ionic compositions were 100% copper ions (39), 100% zinc ions (40), and 50% copper ions / 50% zinc ions (41). The lipid compositions corresponding to the left and right figures are 100% phosphatidylcholine and 60% phosphatidylcholine + 20% phosphatidylglycerol + 20% phosphatidylserine, respectively. Figure 13 The results in (left) indicate that when the phospholipid membrane is entirely composed of phosphatidylcholine, changes in the composition of metal ions do not cause significant differences in the spectrum. This may be because the ion concentration is low (100 μM) at this time, resulting in lower terahertz absorption generated by the ions themselves. Figure 13 In the right image, variations in the composition of metal ions resulted in significant differences in the frequency and amplitude of the metamaterial's resonant peak. In the three experimental groups with different metal ion compositions, the phospholipid composition was consistent, while... Figure 13 The results (left) also indicate that metal ions themselves do not produce a significant optical response, which, combined with the results of [other studies], suggests that metal ions do not produce a significant optical response. Figure 12 Analysis suggests that the differences in the spectra among the three groups originate from different metal ion-membrane interactions. Since different ions have varying affinities for phospholipid membranes, the additional signals generated by metal ion-phospholipid membrane interactions are different. This result demonstrates that the method proposed in this invention can be applied to detect the interactions between mixed metal ions and multi-component phospholipid membranes.

[0110] Example 6: Detecting the complex interactions between multi-component biomembranes and biomolecules such as ions and proteins in artificial cells.

[0111] Example 6 illustrates the use of the method of the present invention to probe the complex interactions between multi-component biomembranes and biomolecules such as ions and proteins in artificial cells. The composition of the natural biomembrane microenvironment is extremely complex, containing not only various glycerophospholipids, sphingomyelins, and sterols, but also a wide variety of interacting biomolecules. These interactions are not isolated; complex relationships exist between different interactions. For example, the binding of metal ions at the biomembrane interface alters the physicochemical properties of the interface, and this alteration may further regulate protein function through membrane-protein interactions. It is this system that accomplishes various complex life activities; therefore, elucidating various interactions simultaneously in artificially constructed model cells is of great significance.

[0112] The strategy proposed in this invention can maintain a near-physiological environment for the sample. For example, water-in-oil reverse micelles are considered by researchers both domestically and internationally as one of the classic "artificial cell" models, which can be used to load various biomolecules and simulate their interactions under physiological conditions. Different interactions alter the dielectric constant of the system to varying degrees, and theoretically, they can be detected separately. In this embodiment, it is important to emphasize that different interactions can be detected independently and without interference. Therefore, this embodiment constructs a near-physiological environment for the sample. Figure 14 The system was used in the experiment. The phospholipid component contained 60% phosphatidylcholine, 20% phosphatidylglycerol, and 20% phosphatidylserine. The biomolecules included copper ions, zinc ions, and bovine serum albumin (BSA). The total concentration of metal ions was 100 μM, and the concentration of BSA was 1 mg / ml. A dual-strip terahertz metamaterial was used to improve detection sensitivity. The specific sample preparation process was the same as in Example 5.

[0113] In this embodiment, terahertz spectra were detected for ion compositions of 100% copper ions 33, 100% zinc ions 34, and 50% copper ions / 50% zinc ions 35. Figure 15 Although the sample contained serum proteins at concentrations similar to those under physiological conditions, the measured terahertz spectrum showed a consistent shift in resonance peak frequency, similar to that in Example 5. As analyzed in Example 5, the main reason for the terahertz spectral changes is the different additional terahertz signals generated by the interactions of different metal ions with the phospholipid membrane. This experimental result demonstrates that interactions between various metal ions and the biomembrane can still be detected even when additional serum proteins interact with the biomembrane in the system. This detection result provides experimental evidence for the detection of complex interactions between multi-component biomembranes and biomolecules such as ions and proteins, as proposed in Example 6, further highlighting the enormous potential of the strategy proposed in this invention in the field of membrane science research.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein, without departing from the concept, spirit and scope of the present invention; and all such modifications or substitutions fall within the scope of protection claimed in the claims of the present invention.

Claims

1. A method for detecting the interaction between biological membranes and biomolecules based on terahertz technology, characterized in that, Includes the following steps: Step 1: Prepare a sample containing the target biomembrane and the target biomolecule; Step 2: Provide a terahertz spectroscopy device, which is equipped with a container for holding the sample described in Step 1 for terahertz detection; Step 3: Add the sample described in Step 1 to the container described in Step 2, and perform terahertz spectroscopy to obtain the optical properties of the terahertz frequency of the interaction between the biological membrane and biomolecules. The optical properties are calculated using the following method: For transmission mode measurements, the absorption coefficient α(ν) at frequency ν is calculated based on the sample detection cavity length d, the terahertz signal intensity Ir(ν) of the empty cuvette, and the terahertz signal intensity Is(ν) transmitted through the sample: ； The refractive index n(ν) is calculated based on the terahertz signal phase Φr(ω) passing through the empty cuvette and the terahertz signal phase Φs(ω) transmitted through the sample: ； The imaginary part of the refractive index, k(ν), is calculated based on the following formula: ； The real part ε'(ν) and the imaginary part ε'(ν) of the dielectric constant are calculated based on the following formula: ； ； For reflection mode measurements, the complex permittivity of the sample Fresnel reflectance based on prism-sample layer 12 Calculation of the terahertz signal incident angle θ and the complex permittivity ε1 of the ATR prism: ； Among them, the Fresnel reflectance coefficient of the prism-sample layer 12 Based on the detected reflectance Phase spectrum Fresnel reflectance of prism-air layer REF calculate: ； ， In step 1, the biomembrane is a self-assembled structure formed by lipid molecules in an aqueous solution. The target biomolecules interacting with the biomembrane are dissolved in the solution. The self-assembly can take the form of either a solid support membrane or a liposome. In step 2, the terahertz spectroscopy device is used to detect samples in either reflection mode or attenuated total reflection mode. In step 1, the biomolecules are a mixture of various metal ions, and the lipids are phosphatidylcholine, phosphatidylglycerol, and phosphatidylserine.

2. The method according to claim 1, characterized in that, The biomolecules mentioned in step 1 are one or more of the following: ions, polypeptides, proteins, nucleic acids, and carbohydrates.

3. The method according to claim 1, characterized in that, The terahertz spectroscopy device mentioned in step 2 is a terahertz time-domain spectrometer or a Fourier transform infrared spectrometer.

4. The method according to claim 1, characterized in that, The container mentioned in step 2 is an optical cuvette or an attenuating total internal reflection prism.

5. The method according to claim 4, characterized in that, The optical cuvette is made of a material with good transmittance in the terahertz band; the cuvette is made of quartz.

6. The method according to claim 1, characterized in that, The terahertz spectroscopy device described in step 2 is also equipped with a terahertz metamaterial sensor.

7. The method according to claim 6, characterized in that, The terahertz material sensor is made of terahertz metamaterials.

8. The method according to claim 6, characterized in that, The terahertz metamaterial is etched with a periodic structure of Asia-Pacific terahertz wavelengths.

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